Ethanol disrupts hepatic function with the eventual appearance of alcoholic liver disease. Kupffer cells, the resident macrophages in the liver, are critical to the onset of ethanol-induced liver injury. Activation of macrophages by endotoxin/ lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, via the toll-like receptor 4 (TLR-4) leads to the production of a variety of inflammatory mediators, such as tumor necrosis factor-1 (TNF-1) and reactive oxygen species. During chronic ethanol exposure, abnormal production of TNF-1 is a critical component in the development of inflammation in the liver. Increased TNF-1 production during ethanol exposure is due to both an increased exposure to LPS, as well as a sensitization Kupffer cells to activation by LPS. This ethanol-induced sensitization results from changes in the regulation of TLR-4 signal transduction, culminating in the dysregulation of transcriptional and post-transcriptional control of TNF-1 expression. The long- term goals of this research project are to understand the molecular mechanisms by which chronic ethanol sensitizes Kupffer cells to TLR-4 mediated signalling and then to leverage this novel information to develop therapeutic strategies to normalize hepatic inflammation in alcoholic liver disease. In the past granting period, we have characterized the molecular mechanisms for ethanol-induced sensitization of LPS-stimulated TNF-1 expression, focusing TLR-4-mediated activation of p38 MAPK and regulation of mRNA stability. More recently, we have investigated the interaction between adiponectin, a potent anti-inflammatory adipokine, and Kupffer cells during chronic ethanol exposure. Treatment of Kupffer cells with adiponectin normalizes the increased sensitivity to LPS in Kupffer cells isolated from ethanol-fed rats. Importantly, Kupffer cells from ethanol-fed rats exhibited increased sensitivity to the anti-inflammatory effects of adiponectin. The major objective of this proposal is to investigate the molecular mechanisms for the anti-inflammatory effects of adiponectin. Utilizing primary cultures of Kupffer cells from ethanol- and pair-fed rats, as well as an in vivo model of LPS challenge in ethanol- and pair-fed mice, the Specific Aims of this proposal will test the following hypotheses: 1) The anti-inflammatory effects of adiponectin on Kupffer cells are due to increased expression of the anti-inflammatory cytokine, IL-10. Further, we hypothesize that adiponectin-stimulated IL-10 production will be higher in Kupffer cells from ethanol-fed rats compared to pair-fed controls. 2) Desensitization of LPS-stimulated signal transduction by adiponectin is due, at least in part, to a normalization of LPS-stimulated NADPH oxidase activity in Kupffer cells from ethanol-fed rats. 3) Adiponectin suppresses/normalizes LPS-stimulated TNF-1 expression in Kupffer cells via transcriptional and post- transcriptional regulatory mechanisms, involving epigenetic regulation and control of mRNA stability, respectively. Understanding the molecular mechanisms for the anti-inflammatory effects of adiponectin in Kupffer cells after chronic ethanol exposure will enable the design of therapeutic strategies aimed at slowing or reversing the progression of alcoholic liver disease. PUBLIC HEALTH RELEVANCE: Alcohol abuse is a leading cause of morbidity and mortality worldwide and recent data indicate that alcoholic liver disease affects over 10 million Americans. The long-term goals of this research project are to investigate the mechanisms by which ethanol disrupts the activity of Kupffer cells, the resident macrophage in the liver. In this application, we will focus on understanding the mechanisms by which the anti-inflammatory mediator, adiponectin, normalizes the sensitivity of Kupffer cells from ethanol-fed animals to stimulation by lipopolysaccharide, a component of gram-negative bacteria. Understanding the mechanisms by which chronic ethanol enhances the inflammatory activity of Kupffer cells will provide the foundation for the future development of rationally designed therapeutic interventions to slow and/or reverse alcoholic liver disease.